Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase.

The Journal of Biological Chemistry
R TirumalaiArthur Landy

Abstract

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical cont...Continue Reading

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Citations

Nov 22, 2001·Biochemical and Biophysical Research Communications·J S YakisichM Cruz
Mar 21, 2002·Proceedings of the National Academy of Sciences of the United States of America·Jonathan M WojciakRobert T Clubb
Mar 16, 2007·The Journal of Biological Chemistry·Danièle MontaudonPhilippe Pourquier
Jun 25, 2015·Microbiology Spectrum·Arthur Landy

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